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PubMed1999

Exo-rhodopsin: a novel rhodopsin expressed in the zebrafish pineal gland.

The zebrafish, a useful animal model for genetic studies, has a photosensitive pineal gland, which has an endogenous circadian pacemaker entrained to environmental light-dark cycles [G.M. Cahill, Brain Res. 708 (1996) 177-181]. Although pinopsin has been found in the pineal glands of birds and reptiles, the molecular identity responsible for fish pineal photosensitivity remains unclear. This study reports identification of a novel opsin gene expressed in the zebrafish pineal gland. The deduced amino acid sequence is similar to, but not identical (74% identity) with that of canonical rhodopsin in the zebrafish retina. This novel rhodopsin is expressed in the majority of pineal cells but not in retinal cells, and hence named exo-rhodopsin after extra-ocular rhodopsin. This study first shows that two different rhodopsin genes are expressed in an individual animal each within a unique location. A phylogenetic analysis indicated that the exo-rhodopsin gene was produced by a duplication of the rhodopsin gene at an early stage in the ray-finned fish lineage. As expected, the exo-rhodopsin gene was found in the medakafish and European eel genomes, suggesting strongly that exo-rhodopsin is a pineal opsin common to teleosts. Identification of exo-rhodopsin in the zebrafish provides an opportunity for studying the role of pineal photoreceptive molecules by using genetic approaches.

Amino Acid Sequence↗
PubMed1998

Mutagenesis studies of human red opsin: trp-281 is essential for proper folding and protein-retinal interactions.

Human red and green opsins contain a strikingly large number of tryptophan residues. These tryptophans are highly conserved among all red and green opsins. To investigate possible roles of these tryptophans in folding and structure, we have systematically replaced each tryptophan of human red opsin. When expressed in COS cells, wild-type red opsin undergoes N-linked glycosylation, forms a light-sensitive pigment with absorption maximum at 560 nm upon reconstitution with 11-cis-retinal, and is transported to the plasma membrane. We used the extent of glycosylation, pigment generation, and intracellular localization of mutant red opsins as our criteria for assessing the effect of substitution. Replacement of eight tryptophans, Trp-59, Trp-90, Trp-149, Trp-152, Trp-183, Trp-191, Trp-195, and Trp-243, with Phe or Ala did not affect the wild-type phenotype significantly. However, replacement of Trp-5 and Trp-51 in the putative N-terminal domain and Trp-142, Trp-177, Trp-179, and Trp-281 in the transmembrane domain with Phe had profound effects, indicating that these substitutions affected red opsin folding. Judged by the severity of the effects, we propose that Trp-5, Trp-51, Trp-177, and Trp-281 are important for red opsin folding. Although substitution of Trp-281 with Phe and Cys did not permit normal glycosylation and transport, substitution with Tyr and His permitted these processes but resulted in blue-shifted pigment. Thus, polar aromatics appear to substitute for Trp-281 to allow red opsin folding. The large spectral shift indicates that Trp-281 is essential for the proper interaction of the protein with 11-cis-retinal.

Amino Acid Sequence↗
PubMed2001

Synthesis of the all-trans-retinal chromophore of retinal G protein-coupled receptor opsin in cultured pigment epithelial cells.

Light-dependent production of 11-cis-retinal by the retinal pigment epithelium (RPE) and normal regeneration of rhodopsin under photic conditions involve the RPE retinal G protein-coupled receptor (RGR) opsin. This microsomal opsin is bound to all-trans-retinal which, upon illumination, isomerizes stereospecifically to the 11-cis isomer. In this paper, we investigate the synthesis of the all-trans-retinal chromophore of RGR in cultured ARPE-hRGR and freshly isolated bovine RPE cells. Exogenous all-trans-[(3)H]retinol is incorporated into intact RPE cells and converted mainly into retinyl esters and all-trans-retinal. The intracellular processing of all-trans-[(3)H]retinol results in physiological binding to RGR of a radiolabeled retinoid, identified as all-trans-[(3)H]retinal. The ARPE-hRGR cells contain a membrane-bound NADPH-dependent retinol dehydrogenase that reacts efficiently with all-trans-retinol but not the 11-cis isomer. The NADPH-dependent all-trans-retinol dehydrogenase activity in isolated RPE microsomal membranes can be linked in vitro to specific binding of the chromophore to RGR. These findings provide confirmation that RGR opsin binds the chromophore, all-trans-retinal, in the dark. A novel all-trans-retinol dehydrogenase exists in the RPE and performs a critical function in chromophore biosynthesis.

Animals↗
PubMed2002

Signaling states of rhodopsin. Formation of the storage form, metarhodopsin III, from active metarhodopsin II.

Vertebrate rhodopsin consists of the apoprotein opsin and the chromophore 11-cis-retinal covalently linked via a protonated Schiff base. Upon photoisomerization of the chromophore to all-trans-retinal, the retinylidene linkage hydrolyzes, and all-trans-retinal dissociates from opsin. The pigment is eventually restored by recombining with enzymatically produced 11-cis-retinal. All-trans-retinal release occurs in parallel with decay of the active form, metarhodopsin (Meta) II, in which the original Schiff base is intact but deprotonated. The intermediates formed during Meta II decay include Meta III, with the original Schiff base reprotonated, and Meta III-like pseudo-photoproducts. Using an intrinsic fluorescence assay, Fourier transform infrared spectroscopy, and UV-visible spectroscopy, we investigated Meta II decay in native rod disk membranes. Up to 40% of Meta III is formed without changes in the intrinsic Trp fluorescence and thus without all-trans-retinal release. NADPH, a cofactor for the reduction of all-trans-retinal to all-trans-retinol, does not accelerate Meta II decay nor does it change the amount of Meta III formed. However, Meta III can be photoconverted back to the Meta II signaling state. The data are described by two quasi-irreversible pathways, leading in parallel into Meta III or into release of all-trans-retinal. Therefore, Meta III could be a form of rhodopsin that is stored away, thus regulating photoreceptor regeneration.

Animals↗
PubMed2006

Evolution of the cichlid visual palette through ontogenetic subfunctionalization of the opsin gene arrays.

The evolution of cone opsin genes is characterized by a dynamic process of gene birth and death through gene duplication and loss. However, the forces governing the retention and death of opsin genes are poorly understood. African cichlid fishes have a range of ecologies, differing in habitat and foraging style, which make them ideal for examining the selective forces acting on the opsin gene family. In this work, we present data on the riverine cichlid, Oreochromis niloticus, which is an ancestral outgroup to the cichlid adaptive radiations in the Great African lakes. We identify 7 cone opsin genes with several instances of gene duplication. We also characterize the spectral sensitivities of these genes through reconstitution of visual pigments. Peak absorbances demonstrate that each tilapia cone opsin gene codes for a spectrally distinct visual pigment: SWS1 (360 nm), SWS2b (423 nm), SWS2a (456 nm), Rh2b (472 nm), Rh2a beta (518 nm), Rh2a alpha (528 nm), and LWS (561 nm). Furthermore, quantitative reverse transcription polymerase chain reaction at 3 ontogenetic time points demonstrates that although only 4 genes (SWS2a, Rh2a alpha and beta, and LWS) are expressed in adults, mRNAs for the other genes are all expressed during ontogeny. Therefore, subfunctionalization through differential ontogenetic expression may be a key mechanism for preservation of opsin genes. The distinct peak absorbances of these preserved opsin genes provide a palette from which selection creates the diverse visual sensitivities found among the cichlid species of the lacustrine adaptive radiations.

Animals↗
PubMed1996

High-level inducible expression of visual pigments in transfected cells.

A method for high-level expression of a functionally active, recombinant human red cone opsin was developed by adding the coding sequence for the C-terminal epitope of bovine rhodopsin onto the C terminus of the cone opsin and cloning the resulting construct into the vector pMEP4 beta. The recombinant pMEP4 beta vector was transfected stably into 293-EBNA cells, and expression of the cone opsin was induced by the addition of CdCl2 into the medium. The recombinant cone opsin was reconstituted with 11-cis retinal and purified by immunoaffinity chromatography. Spectral analysis prior to and following photobleaching confirmed its identity as a red cone opsin. The protein was targeted to the cell membrane and activated bovine transducin.

Animals↗
PubMed1990

The eye of the blind mole rat, Spalax ehrenbergi. Rudiment with hidden function?

The rudimentary eyes of the mole rat (Spalax ehrenbergi) are located under the skin and do not respond to light stimuli. However, removal of the eyes disturbs photoperiod perception in these animals. To help clarify the possibly remaining function of the eyes in this species, the authors studied their development and ultrastructure. In the early embryos the presumptive eye regions--the epithelium, lens vesicle, and optic cup--appear initially normal. As development progresses, the iris-ciliary body complex originates prematurely from the margin of the optic cup and shows a very rapid and massive growth. This pigment-laden tissue mass remains attached to the corneal stroma, obliterates the anterior chamber, and prevents the formation of the corneal endothelium and Descemet's membrane. In the developing lens the elongation of the lens fibers leads to the formation of a rudimentary lens nucleus that becomes disorganized and vacuolated and eventually also becomes vascularized. The optic fissure fails to close, the eyes remain colobomatous, and the optic disc appears atrophic. In contrast, retinal histogenesis progresses relatively normally, resulting in structurally reduced but well-differentiated photoreceptor, neuronal, and ganglion cell layers in the adult eye. Immunohistochemically, the presence of opsin could be demonstrated in the photoreceptor cells. The latter features may indicate that these rudimentary eyes are still functioning in the complex neuroendocrine pathways mediating photoperiodicity.

Animals↗
PubMed1990

Orientation of retinal in bovine rhodopsin determined by cross-linking using a photoactivatable analog of 11-cis-retinal.

A photoactivatable analog of 11-cis-retinal has been used to probe the orientation of retinal in bovine rhodopsin. The analog binds to the opsin to regenerate a chromophore with lambda max at 458 nm. The linkage site of the analog to the opsin was confirmed to be Lys-296 as in 11-cis-retinal rhodopsin. The analog-reconstituted rhodopsin activated transducin and was phosphorylated by rhodopsin kinase on illumination. On photolysis of rhodopsin containing the radioactively labeled analog at 365 nm at -15 degrees C, 20-25% of the analog was covalently linked to the protein. Proteolysis of the labeled protein and characterization of the appropriate peptides showed that cross-linking of the analog was predominantly to helices C or F. When analog reconstituted rhodopsin in rod outer segments was photolyzed, cross-linking was predominantly to helix C. However, when analog-reconstituted rhodopsin, purified in lauryl maltoside, was photolyzed, labeling occurred mainly in helix F. Sequence analysis showed major sites of cross-linking to be Phe-115, Ala-117, Glu-122, Trp-126, and Ser-127 in helix C while Trp-265 was the major site in helix F. The results suggest that the beta-ionone ring of retinal orients toward helices C and F.

Amino Acid Sequence↗
PubMed1997

Short wavelength-sensitive opsins from the Saharan silver and carpenter ants.

We have previously cloned the opsins coding for the long-wavelength visual pigments from the Saharan silver ant and carpenter ant. Here we report two new cDNA clones isolated from cDNA libraries which also code for opsin proteins. These cDNAs code for deduced proteins with 369 amino acids which are 91% identical to each other, but only 38% identical to the previously cloned opsins. Phyletic comparisons suggest that these opsins are likely the ultraviolet sensitive visual pigments, a conclusion that is supported by the presence of a phenylalanine at the counterion position in the third transmembrane segment.

Animals↗
PubMed1993

Maturation of major Drosophila rhodopsin, ninaE, requires chromophore 3-hydroxyretinal.

Opsin expression is extremely suppressed by carotenoid deprivation in Drosophila. Carotenoid replacement in deprived flies promotes the recovery of visual pigment with an increase in opsin, as well as the chromophore 11-cis-3-hydroxyretinal. Here, we show that opsin mRNA and opsin peptide in an intermediate step of posttranslational processing were present in carotenoid-deprived flies. By supplementing chromophore to photoreceptor cells, intermediate opsin was made mature. During this process, opsin peptide underwent multiple modifications involving glycosylation. Based on these results, we present a novel mechanism of protein regulatory expression; that is, chromophore posttranslationally controls the expression of apoprotein by promoting its maturation.

Animals↗
PubMed2003

Molecular evolution of bat color vision genes.

The two suborders of bats, Megachiroptera (megabats) and Microchiroptera (microbats), use different sensory modalities for perceiving their environment. Megabats are crepuscular and rely on a well-developed eyes and visual pathway, whereas microbats occupy a nocturnal niche and use acoustic orientation or echolocation more than vision as the major means of perceiving their environment. In view of the differences associated with their sensory systems, we decided to investigate the function and evolution of color vision (opsin genes) in these two suborders of bats. The middle/long wavelength (M/L) and short wavelength (S) opsin genes were sequenced from two frugivorous species of megabats, Haplonycteris fischeri and Pteropus dasymallus formosus, and one insectivorous species of microbat, Myotis velifer. Contrary to the situation in primates, where many nocturnal species have lost the functional S opsin gene, both crepuscular and strictly nocturnal species of bats that we examined have functional M/L and S opsin genes. Surprisingly, the S opsin in these bats may be sensitive to UV light, which is relatively more abundant at dawn and at dusk. The M/L opsin in these bats appears to be the L type, which is sensitive to red and may be helpful for identifying fruits among leaves or for other purposes. Most interestingly, H. fischeri has a recent duplication of the M/L opsin gene, representing to date the only known case of opsin gene duplication in non-primate mammals. Some of these observations are unexpected and may provide insights into the effect of nocturnal life on the evolution of opsin genes in mammals and the evolution of the life history traits of bats in general.

Amino Acid Sequence↗
PubMed1997

A new rhodopsin in R8 photoreceptors of Drosophila: evidence for coordinate expression with Rh3 in R7 cells.

The photoreceptor cells of the Drosophila compound eye are precisely organized in elementary units called ommatidia. The outer (R1-R6) and inner (R7, R8) photoreceptors represent two physiologically distinct systems with two different projection targets in the brain (for review see Hardie, 1985). All cells of the primary system, R1-R6, express the same rhodopsin and are functionally identical. In contrast, the R7 and R8 photoreceptors are different from each other. They occupy anatomically precise positions, with R7 on top of R8. In fact, there are several classes of R7/R8 pairs, which differ morphologically and functionally and are characterized by the expression of one of two R7-specific opsins, rh3 or rh4. Here, we describe the identification of a new opsin gene, rhodopsin 5, expressed in one subclass of R8 cells. Interestingly, this subclass represents R8 cells that are directly underneath the R7 photoreceptors expressing rh3, but are never under those expressing rh4. These results confirm the existence of two subpopulations of R7 and R8 cells, which coordinate the expression of their respective rh genes. Thus, developmental signaling pathways between R7 and R8 lead to the exclusive expression of a single rhodopsin gene per cell and to the coordinate expression of another one in the neighboring cell. Consistent with this, rh5 expression in R8 disappears when R7 cells are absent (in sevenless mutant). We propose a model for the concerted evolution of opsin genes and the elaboration of the architecture of the retina.

Amino Acid Sequence↗
PubMed1982

Immunoreactive opsin in the pineal organ of reptiles and birds.

The presence of opsin was investigated with light microscopic immunocytochemistry in pinealocytes of reptiles and birds (Emys orbicularis, Pseudemys scripta elegans, Lacerta agilis et viridis, Gallus domesticus, Columba livia, Melopsittacus undulatus, Serinus canaria, Taeniopyga punctate). The outer segments of pinealocytes selectively bound antiopsin antibody as revealed by indirect immunocytochemical techniques, indicating the occurrence of a rhodopsin-like photopigment in these structures. The results were compared with those obtained in retinal photoreceptors of the same species as well as in the pineal organ of fishes and amphibians (Cyprinus carpio, Carassius auratus, Rana esculenta). Corresponding to immunoreactive structures seen in the light microscope, we found typical outer segments on a large number of pinealocytes in most of the reptiles and birds studied. The presence of opsin in the numerous well developed pineal outer segments of these reptilian and avian species contradicts the earlier hypothesis on the gradual regression of pineal sensitive structures in the avian line of evolution.

Animals↗
PubMed1997

A new form of inherited red-blindness identified in zebrafish.

A red-blind zebrafish mutant, partial optokinetic response b (pob), has been isolated by measuring eye movements of larvae in a three-generation screen for recessive mutations affecting the visual system. pob larvae exhibit eye movements in response to rotating black and white stripes illuminated with white light, but they do not move their eyes when the stripes are illuminated with red light. Physiological, immunohistochemical, and in situ hybridization analyses of pob retinas showed a selective loss of red-sensitive cones at 5 days postfertilization (dpf). At 3 dpf, cells expressing red opsin are present, suggesting that red-sensitive cones form initially but then disappear rapidly, whereas other photoreceptors remain. Linkage analysis indicated that the mutation identified in the pob mutant is not at the red opsin locus. Because red opsin is the only known molecule unique to red cones, these data suggest that a novel gene is required for the maintenance or function of red cones.

Animals↗
PubMed2001

Molecular cloning of Bombyx cerebral opsin (Boceropsin) and cellular localization of its expression in the silkworm brain.

We have cloned a cDNA for a novel opsin from the larval brain of the silkworm Bombyx mori in which the photoperiodic photoreceptor had been supposed to reside in the cephalic central nervous system (CNS). Its deduced amino acid sequence was composed of 381 amino acids and included amino acid residues highly conserved in insect visual pigments. This opsin belonged to the long wavelength photoreceptor group of insect opsins and showed the greatest degree of homology (84%) with the green visual photoreceptor in the sphingid moth. We have designated this Bombyx cerebral opsin as Boceropsin. Southern blotting experiments indicated that the Boceropsin gene is present in a single copy, and RT-PCR analysis revealed that Boceropsin mRNA is expressed in the larval brain but not in the subesophageal ganglion (Sg) or thoracic ganglion (Tg). Immunohistochemical analyses demonstrated that Boceropsin protein is present bilaterally in some defined cells localized in the brain of Bombyx larvae. This is the first report of expression of an opsin-based protein in CNS of an insect. The possibility that the Boceropsin functions as the photoperiodic receptive pigment in the silkworm is also discussed.

Amino Acid Sequence↗
PubMed2001

Microenvironmental regulation of visual pigment expression in the chick retina.

Visual pigment (VP) expression in the chick embryo retina was investigated in ovo, in dissociated and explant cultures, and in cDNAs from individual cells. While VP mRNA is not detectable by in situ hybridization until embryonic day (ED) 14-16 in ovo, analysis of VP expression by RT-PCR showed that VP messages are present in the retina as many as 7-10 days before they become detectable by in situ hybridization, and are also detected in other regions of the embryonic CNS. On the other hand, red opsin expression is markedly accelerated when cells are isolated from their intraocular microenvironment at ED 6, and placed in pigment epithelium-free dissociated or explant cultures. This acceleration occurs regardless of cell density, birth date, or serum presence in the medium, suggesting that many photoreceptors are already programmed to express red opsin on or before ED 6, and that microenvironmental inhibitory factors prevent implementation of this program until ED 14 in ovo. The selectivity of this phenomenon is suggested by the finding that other VPs are not observed by in situ hybridization in ED 6 cultures, although they are detectable in cultures of older retinas. Taken together, these findings suggest that red opsin expression may be constitutive for many developing photoreceptor cells in the chick.

Animals↗
PubMed1995

Two visual pigment opsins, one expressed in the dorsal region and another in the dorsal and ventral regions, of the compound eye of a dragonfly, Sympetrum frequens.

This paper describes the primary structure of two visual pigment opsins (DfRh1 and DfRh2) in the regionalized compound eye of a dragonfly, Sympetrum frequens. The amino acid sequences were deduced from the nucleotide sequences of cDNAs isolated from a cDNA library of the dragonfly retina. The two opsins both consist of 379 amino acids with 81.3% identity. Analysis of hydropathy indicated that the sequences have seven transmembrane domains like those of previously described opsins. Expression analysis using RT-PCR revealed that DfRh1 was present only in the dorsal region whereas DfRh2 was detected in both the dorsal and the ventral regions of the eye.

Amino Acid Sequence↗
PubMed1989

Wavelength regulation in iodopsin, a cone pigment.

The opsin shift, the difference in wavenumber between the absorption peak of a visual pigment and the protonated Schiff base of the chromophore, represents the influence of the opsin binding site on the chromophore. The opsin shift for the chicken cone pigment iodopsin is much larger than that for rhodopsin. To understand the origin of this opsin shift and the mechanism of wavelength regulation in iodopsin, a series of synthetic 9-cis and 11-cis dehydro- and dihydro-retinals was used to regenerate iodopsin-based pigments. The opsin shifts of these pigments are quite similar to those found in bacteriorhodopsin-based artificial pigments. On the basis of these studies, a tentative model of wavelength regulation in iodopsin is proposed.

Animals↗